Mycoplasma pneumoniae is the most significant of the mycoplasmal human pathogens, causing 20% to 30% of all community-acquired pneumonia. Even in mild cases, the economic impact of infection is considerable due to disease chronicity and associated loss of work. Serious extrapulmonary complications are not uncommon and include encephalitis and meningitis. The organism may also play a role in the pathogenesis of asthma. Mycoplasma pulmonis is a natural pathogen of rats and mice, the causative agent of murine respiratory mycoplasmosis (MRM). MRM shares many pathologic features with M. pneumoniae infection and is by far the best-characterized animal model of mycoplasmal disease. We believe that M. pulmonis and M. pneumoniae both produce at least two polysaccharides that are critical for pathogenesis. One of the polysaccharides may be capsular and the other required for a biofilm that can form in vivo and confer resistance to host defenses and antibiotic therapy. Although they have been virtually ignored by the field of mycoplasmology for the past 30 years, polysaccharides likely contribute to the pathogenesis and chronicity of disease as they do for most pulmonary bacterial pathogens. As such, the polysaccharides or the machinery that produces them, are potential therapeutic targets. The production of polysaccharides by these organisms is perhaps surprising because genome annotations identify very few genes with a potential role in polysaccharide synthesis. Mutants of M. pulmonis that fail to produce the polysaccharide EPS-I or the polysaccharide EPS-II have been identified and have transposon disruptions in genes that resemble ABC transporters and not conventional polysaccharide synthesis machinery. Polysaccharide synthesis in M. pulmonis may be novel and additional evidence suggests that the substrate for synthesis may be something other than nucleotide sugars. Whether polysaccharide synthesis in M. pneumoniae is similar to that of M. pulmonis remains to be determined. Aim 1 of this proposal is to further study polysaccharide synthesis in M. pulmonis through the characterization of existing mutants and the isolation of additional mutants. The goal is to identify the glycosyltransferases and substrates used for polysaccharide synthesis. One approach is to identify the substrates, and/or the intermediates in the pathway of substrate synthesis, that are missing or overproduced in the mutants. A second approach is to identify reaction conditions that support polysaccharide synthesis in vitro. A third is to clone the genes required for polysaccharide synthesis into other bacteria for study. Aim 2 is to translate what is learned in M. pulmonis to M. pneumoniae. Mutants of M. pneumoniae that fail to produce one or more polysaccharides will be isolated and studied as described for aim 1. The completion of these aims will have a major impact by establishing mycoplasmal polysaccharides as virulence factors and targets for control of infection.